Chymosin is an aspartic proteinase found in the stomach of neonatal mammals and is used as milk coagulant in the cheese industry. In this study, preprochymosin cDNA from the abomasums of Iranian natives’ kid goats was cloned and characterized. This cDNA has an open reading frame of 1143 bp and is predicted to code for a preproenzyme of 381 amino acids with an N-terminal 16 amino acid signal peptide that is followed by a 42 amino acid proenzyme. The deduced amino acid sequence revealed 98.7% and 94% identity with corresponding lamb and cattle sequences, respectively. The cDNA encoding for prochymosin was then subcloned into pET-28a and expressed with and without N-terminal His-tag in E. coli BL21 (DE3). Inclusion bodies were isolated and solubilized in 8 M urea at pH 10.7. Solubilized prochymosion molecules were successfully refolded and active recombinant goat chymosin was recovered after subsequent activation. Milk clotting activity (416 U/mg and 192 U/mg) was observed in (His+)-prochymosin and (His- )-prochymosin enzymes, respectively, after activation.
کلید واژگان :caprine; recombinant prochymosin; His-tag; activity
ارزش ریالی : 600000 ریال
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